Project Summary Infectious diseases impose a significant burden on human health, with epithelial cells on the front lines of attack by disease-causing microbes that can invade and replicate inside of these cells. In addition to diseases such as diarrhea and pneumonia caused by pathogens, inappropriate activation of defense pathways in epithelial cells can lead to inflammatory disease. Therefore, it is critical learn more about epithelial defense against intracellular pathogens. In particular, little is known about defense against the Microsporidia phylum of fungal-related intracellular parasites, 14 of which can infect and cause disease in humans, most commonly infecting intestinal epithelial cells. We have developed a convenient whole-animal model for studying defense against microsporidia through characterization of natural intestinal infection in the nematode C. elegans. Our long-term goal is to dissect the mechanisms by which epithelial cells defend against co-evolved intracellular pathogens like microsporidia. Closing this gap in our understanding will provide new insights for treating infectious disease and inflammatory disorders. Our central hypothesis is that attack from co-evolved pathogens causes expansion and diversification of host genes to become ?species-specific? although these genes may control conserved immune pathways. The objective here is to characterize the species-specific pals gene family, which expanded to 39 pals genes in C. elegans, whereas there is only one pals gene in humans. Virtually nothing is known about PALS protein structure or biochemical function in any system, although they have been connected to ubiquitin ligases through sequence analysis and genetic studies in C. elegans. We found PALS-22 and PALS-25 to be key regulators of a common transcriptional response to natural microsporidia and viral infections in C. elegans that we call the Intracellular Pathogen Response or IPR. The IPR appears to define an entire physiological program and our forward genetic screens identified PALS-22 as a negative regulator and PALS-25 as a positive regulator of the IPR. Loss of PALS-22 leads to enhanced immunity against intracellular pathogens, increased RNA interference, as well as fitness consequences such as delayed development, all of which depend on PALS-25. In Specific Aim 1 we will characterize the relationship between gene expression and immune responses regulated by PALS-22 and PALS-25, identify the tissues where they act, and define the stage of microsporidia they target. In Specific Aim 2 we will recombinantly express PALS-22 and PALS-25 proteins to characterize their interaction, as well as their structure using X-ray crystallography and cryo EM. In Specific Aim 3 we will analyze the role of other PALS proteins, RNAi machinery and identify new regulators of the IPR. The approach is innovative as it focuses on uncharacterized proteins that regulate a novel form of epithelial immunity. The proposed research is significant, because it could lead to new treatments for infectious diseases and inflammatory disorders.